Many bioanalytical procedures such as affinity purification and many biochemical assays such as immunoassays and DNA hybridization assays require the separation of specific molecules or constituents from a complex mixture. In the context of molecular and cell biology, magnetizable polymeric particles (“beads”) have been widely used for this aspect of sample preparation. For example, magnetic beads displaying a short oligo-dT capture probe serve to extract messenger RNA (mRNA) molecules from a cell lysate. Following addition of particles to the lysate, molecules are captured by hybridization of their poly-A tails to the capture probe, trapped in a magnetic field gradient generated by a permanent magnet, retained by the applied magnetic field during the exchange of the lysate for fresh buffer and released into suspension by removal of the magnetic field (“Biomagnetic Techniques in Molecular Biology,” Technical Handbook, 3rd Edition, DYNAL, 1998). In a similar manner, magnetic beads displaying antibodies directed against specific cell surface antigens serve to selectively extract cells of the desired type from a given suspension (“Cell Separation and Protein Purification”, Technical Handbook, 2nd edition, DYNAL, 1997). A recent example describes a method of magnetic cell separation describes the use of magnetic particles in conjunction with optical imaging of trapped cells (A. G. J. Tibbe et al. “Optical tracking and detection of immunomagnetically selected and aligned cells” Nature Biotech., 17, 1999, 1210-1213).
The integration of assay steps, a principal objective motivating the introduction of clinical analyzers and other examples of laboratory automation, in today's state of the art relies upon a 96-well (or related) microwell format of multiple discrete reaction wells to accommodate standard robotic liquid handling (“pipetting”) and reading of assay signals from individual wells by plate readers. Commercial robotic pipetting systems have been recently introduced to automate sample preparation based on the use of magnetic beads for separation. However, the integration of sample processing and a highly parallel array format of analysis by way of microfluidic operations, highly desirable in connection with the miniaturization of biochemical and analytical assay procedures, has not been described to date.
The imprinting of multiple binding agents such as antibodies and oligonucleotides on planar substrates in the form of spots or stripes facilitates the simultaneous monitoring of multiple analytes such as antigens and DNA in parallel (“multiplexed”) binding assays. The miniaturization of this array format for increasing assay throughput and studying binding kinetics are described (R. Ekins, F. W. Chu, Olin. Chem. 37, 955-967 (1991); E. M. Southern, U. Maskos, J. K. Elder, Genomics 13, 1008-1017 (1992)). In recent years, this approach has attracted substantial interest particularly in connection with performing extensive genetic analysis (G. Ramsay, Nat. Biotechnol. 16, 40-44 (1998); P. Brown, D. Botstein, Nat. Genet. 21, 33-37 (1999); D. Duggan, M. Bittner, Y. Chen, P. Meltzer, J. M. Trent, Nat. Genet. 21, 10-14 (1999); R. Lipshutz, S. P. A. Fodor, T. R. Gingeras, D. J. Lockhart, Nat. Genet. 21, 20-24 (1999)).
The principal techniques of array fabrication reported to date include: refinements of the original “spotting” in the form of pin transfer or ink jet printing of small aliquots of probe solution onto various substrates (V. G. Cheung, et al., Nat. Genet. 21, 15-19 (1999)); sequential electrophoretic deposition of binding agents in individually electrically addressable substrate regions (J. Cheng, et al., Nat. Biotechnol., 541-546 (1998)), and methods facilitating spatially resolved in-situ synthesis of oligonucleotides (U. Maskos M. Southern, Nucleic Acids Res. 20, 1679-1684 (1992); S. P. A. Fodor, et al., Science 251, 767-773 (1991)) or copolymerization of oligonucleotides (A. V. Vasiliskov, et al., BioTechniques 27, 592-606 (1999)). These techniques produce spatially encoded arrays in which the position within the array indicates the chemical identity of any constituent probe (BioTechniques 27, 592-606 (1999)). All of these techniques of the prior invention require that array formation be completed prior to initiation of the assay of interest. Therefore, none of the techniques of array formation of the prior art permit the real-lime formation of arrays subsequent to completion of the binding interaction of interest.
Monodisperse magnetic particles confined to planar substrates or interfaces, and exposed to a uniform magnetic field oriented normal to the plane of the interface, form a variety of ordered two-dimensional structures (W. Wen, L. Zhang and P. Sheng “Planar Magnetic Colloidal Crystals” Phys. Rev. Lett., 85, (25), 5464-5466, 2000; M. Golosovksy, Y. Saado, and D. Davidov “Self-assembly of floating magnetic particles into ordered structures: A promising route for the fabrication of tunable photonic band gap materials” Appl. Phys. Lett., 75, (26), 4186-4170, (1999); K. Zhan, R. Lenke, and G. Maret “Two-stage melting of paramagnetic colloidal crystals in two dimensions” Phys. Rev. Letter., 82, (13), 2721-2724, 1999).
Many techniques have been suggested for the synthesis of these particles. These techniques attempt to endow the magnetic particles with certain properties that make them desirable for certain applications. These techniques can be grouped into two categories, the first category relating to synthesis of a magnetic core and the second category relates to the synthesis of a magnetic shell.
Patents that may be considered of interest in the first category include:
U.S. Pat. No. 4,358,388 to Daniel et al and U.S. Pat. No. 5,356,713 to Charmot et al. disclose a process which utilizes a suspension polymerization approach. One drawback of the process is the difficulty in controlling the mono-dispersity of the resulting magnetic Latex, and the process does not appear well suited for the generation of fluorescent magnetic particles
U.S. Pat. No. 4,654,267 to Ugelstead et al discloses a nitration method which produces particles with a para-magnetic core. Following magnetization, the particles are coated with functional polymers to provide a reactive shell to produce super-paramagnetic particles of controlled morphology, polydispersity, pore size distribution, magnetic loading and surface chemistry. The encoding of such particles has not been described.
U.S. Pat. No. 4,873,102 to Chang et al discloses a process of forming magnetic polymer particles containing crystals of magnetite uniformly throughout the pores. The particles can only be used under hydrophilic conditions.
U.S. Pat. No. 5,356,713 to Charmot et al discloses magnetizable composite microspheres which are useful in biological applications but are limited by their size distribution to other applications.
U.S. Pat. No. 5,512,439 to Hornes at al discloses monodisperse, super-magnetic particles carrying a plurality of molecules of an oligonucleotide which may be used for single stranded nucleic acids. The oligonucleotides may be covalently attached or affinity bonded.
U.S. Pat. No. 5,698,271 to Liberti discloses a method for the manufacture of magnetically responsive particles. The particles have applications in a variety of preparative and diagnostic techniques.
U.S. Pat. No. 5,866,099 to Owen et al discloses a magnetic-polymer particle useful in immunoassay techniques and biological/medical applications. The particle is produced by co-precipitation of transition metals in the presence of a polymer having available coordination sites.
Patents that may be considered of interest in the second category include:
U.S. Pat. No. 5,736,349 to Sasaki et al discloses a magnetic particle for an immunoassay method which comprises a core and a coating layer. An antigen or antibody is bound onto the surface of the coating layer.
U.S. Pat. No. 5,648,124 to Sutor et al discloses a process for the production of magnetic particles by hetero-coagulation utilizing oppositely charged core particles and magnetite particles. The dispersed magnetite may be a coated microparticle which can be further coated with one or more outer polymeric coatings.
U.S. Pat. Nos. 6,013,531, 5,283,079 and 5,091,206 to Wang et al disclose a process for producing magnetically responsive polymer particles. The particles comprise a polymeric core particles coated evenly with a layer of polymer containing magnetically responsive metal oxide. The surface of these magnetically responsive polymer particles can be coated further with a layer of functionalized polymer. These magnetically responsive polymer particles can be used for passive o covalent coupling of biological material and used as solid phase for various types of immunoassays.
Several methods have been described for the synthesis of stained magnetic particles. Patents that may be considered of interest include U.S. Pat. No. 5,395,688 to Wang which discloses a process for producing magnetically responsive fluorescent polymer particles composed of a fluorescent polymer core particle that is evenly coated with a layer of magnetically responsive metal oxide. The method utilizes preformed fluorescent polymeric core particles which are mixed with an emulsion of styrene and magnetic metal oxide in water and polymerized. A two-step reactive process such as this suffers from the drawback of possible inhibition of polymerization by the fluorescent dye or conversely bleaching of the fluorescence by the shell polymerization process. The use of such magnetic particles containing fluorescent tags for the calibration of certain solid phase assays has been described in U.S. Pat. No. 6,013,531. Following completion of this step, the particles are coated with functional polymers to provide a reactive shell.
The creation of core-shell particles from dispersed colloidal matter can be accomplished by a multistep (layer-by-layer) strategy. The process involves step-wise adsorption of charged polymers or nanoparticles and oppositely charged polyelectrolytes onto colloidal particles, exploiting primarily electrostatic interactions for layer buildup. (Caruso et al “Magnetic Core-Shell Particles: Preparation of Magnetite Multilayers on Polymer Latex Microspheres” Adv. Mater. 1999, 11, 950-953) A shell applied by electrostratic physisorption is not desirable for bioanalytical assays because it is chemically unstable under changes of assay conditions, particularly salt concentration, and promotes non-specific adsorption and denaturation of charged biomolecules; the particles described in this prior art reference are unsuitable in connection with the assay formats contemplated herein.
While the foregoing references disclose the use of magnetic particles, none of the prior art particles appear to possess the properties that are necessary to meet the criteria which are necessary for the successful performance of the assays described herein including a preferred size range, substantial monodispersity, chemical functionalization and synthetic flexibility, the latter permitting the rapid construction of libraries of encoded magnetic particles that can be functionalized on demand, the chemical diversity represented in these libraries greater than 2. In addition, magnetic particles must meet certain standards of quality to permit the reproducible assembly of customized arrays to ensure consistent performance in quantitative assays.